Peptides for use in culture media

ABSTRACT

The present invention provides peptides libraries which are useful for rapid identification of biologically active compounds. The invention further provides peptides which include cell-growth affecting peptides and peptides which enhance or inhibit production of cellular proteins. Many of the peptides of the invention may be produced in large quantity by recombinant techniques and formulated in culture medium to produce the desired effect on cultured cells and tissues. Certain of the libraries of the invention and the peptides identified in them are particularly useful in concatemer-based recombinant expression methods.

FIELD OF THE INVENTION

The invention relates to peptides which affect cells in culture and tomethods for discovery and manufacture of such peptides. In particular,the invention relates to peptides which affect growth of cells inculture and peptides which affect cellular protein production.

BACKGROUND OF THE INVENTION

Tissue and protein hydrolysates have been routinely used as a source ofpeptides in cell culture media since the late 1800's. They are the mostcommon undefined culture media component in present use in bacteriologyand often replace serum in mammalian culture (S. Saha and A. Sen. 1989.Acta Virol. 33:338-343). Hydrolysates and serum are not optimal sourcesof peptides for culture media, however, because their compositions areundefined and variable, and serum may harbor pathogens such as BSE.

It has been recognized that peptides are generally preferred nutrientsas compared to their constituent amino acids. Several approaches havebeen taken in an effort to determine which specific peptides areutilized by a cell culture as a means for identifying defined peptideswhich affect growth or some other biological activity. A common practiceis to analyze spent media in an attempt to identify compounds which havebeen consumed during culture. This seldom leads to a single compoundwhich can be isolated and studied. The spent media approach cannotidentify compounds which affect the cell and are not removed from themedium (e.g., signaling compounds). In an alternative approach, specificproteins are digested and the HPLC-purified peptide fragments are spikedback into the medium to evaluate their effects. This approach mayidentify a peptide which performs better than the whole protein digestor tissue digest, but the number of possible peptides for analysis islimited. For example, it has been reported that casein hydrolyzed by theneutral protease of Micrococcus caseolyticus (M. J. Desmazeaud and J. H.Hermier. 1972. Eur. J. Biochem. 28:190-198) and a papain digest ofglucagon (M. J. Desmazeaud and J. H. Hermier. 1973. Biochimie55:679-684) enhance the growth of Streptococcus thermophilus. In thiscase the stimulatory peptides were isolated and characterized. It hasalso been found that trypsin digested κ-casein enhances the growth ofthe genus Bifidobacterium (M. Poch and A. Bezkorovainy. 1991. J. Agric.Food Chem. 39:73-77), however, the specific peptides which produce thisresult were not identified. Azuma, et al. (1984. Agric. Biol. Chem.48:2159-2162) and Bezkorovainy, et al. (1979. Am. J. Clin. Nutr.32:1428-1432) reported that a glycopolypeptide derived fromenzyme-digested human casein promotes growth of Lactobacillus bifidus.Tryptic fragments of human β-casein have also been reported to stimulateDNA synthesis in BALB/c3T3 cells (N. Azuma, et al. 1989. Agric. Biol.Chem. 53:2631-2634). The sequences of two such tryptic fragments weredetermined. These prior art methods are time consuming and have resultedin identification of only a few peptides which generate marginalimprovements. Their success has primarily been limited by the rawmaterials, which are restricted by the starting substrates and thedigesting agents used in their preparation.

More recently, developments in peptide synthesis technology have made itat least plausible to prepare and screen large numbers of compounds formedia enhancement, either as individual defined sequences or as amixture of variable sequences in a peptide library. Many of thesesequences would not be present or detected in the traditional undefinedmaterials. The library approach has provided an opportunity to screenmore peptide sequences for desired biological effects in cell culture,but the primary methods have major disadvantages. Assaying compoundsindividually means screening millions of samples containingrandomly-generated sequences. As a practical matter, the exhaustivesynthesis and screening of libraries is often prohibitively expensiveand also time-consuming. The combinatorial approach runs the risk ofmissing potential lead compounds due to poor representation (lowconcentration) of each compound in the cocktail due to solubilityrestraints and masking effects that can occur from competing compounds.In an effort to reduce the number of sequences which must be screened ina library, practitioners have “fixed” certain residue positions duringsynthesis of the library. That is, certain residues at certain positionsin the sequence are added randomly, but the residues at other positionsare defined. Such a synthetic peptide library is described in U.S. Pat.No. 5,556,762. In addition, Geysen (WO 86/00991) describes librariescomprising peptide sequences which are a combination of defined andundefined amino acid residues. Furka, et al. (1988. 14^(th) Int. Cong.Biochem. Vol. 5, Abst. FR:013) discloses relatively simple mixtures oftetrapeptides in which the N- and C-terminal residues are fixed and anyone of three residues occur at each position in between. Fodor, et al.(1991. Science 251:767-773) teach solid phase peptide synthesis onslides, using predetermined amino acids coupled to defined areas of theslide using photomasks. In this way an array of 1024 different peptideswith defined C-termini was synthesized. All of these techniques attemptto circumvent the individual screening of millions of peptides and toincrease the amount of a given sequence in the library to simplifyscreening and identification of biologically active peptides.

While fixed-position (i.e., limited diversity) libraries reduce thenumber of sequences which must be screened, they also limit the numberof different sequences available for screening and thus may reduce theprobability of identifying a sequence with the desired properties. Inthe publications discussed above, there was typically no attempt made to“re-expand” the number of available sequences in order to identifyadditional sequences which may have properties similar to those in thelimited diversity library. Recently, information about the properties ofa compound identified in a more limited library of sequences has beenused to generate a more diverse library of compounds which arestructurally similar to the initial compound identified. Theseadditional compounds, which were not present in the initial library, mayexhibit biological activities which are similar to the initial leadcompound. This approach is often referred to as rational design oftargeted libraries. See, for example, S. Cho, et al. 1998. J. Chem. Inf.Comput. Sci. 38:259-268.

For media applications, simply identifying a compound delivering thedesired enhancement is not sufficient. To impact the overall mediaoptimization process, a lead compound must be rapidly scaled up and madeavailable in a time frame which will impact the typical mediaoptimization cycle. Further, the method used for the initial scale upmust be in-line with the planned commercial manufacturing processcapable of delivering the compound at a cost in-line with benefit. Theideal discovery process would link the initial library design to thepreferred manufacturing process and thereby avoid a series of subsequentlibraries aimed at finding compounds with similar performance attributesthat can also be manufactured. None of the existing fixed-librarydesigns address this need. Indeed, the manufacturing aspect is left tochance.

There is therefore a need in the art for chemically-defined peptideswith well-characterized biological activities which can be added toculture media to produce a desired biological effect. Such peptidesreduce the number and quantity of undefined components in culture media,reduce the need for animal-derived components, improve media consistencyand quality control and provide a means for precisely controlling andadjusting performance of the cell culture. The present invention employsa peptide library approach to select and identify peptides which meetthese needs, in particular a process that links discovery andmanufacturing of peptides which affect cell growth (either positively ornegatively) or which enhance or inhibit cellular protein production.

SUMMARY OF THE INVENTION

The present invention provides peptide libraries which are useful forrapid identification of biologically active compounds which affect theproperties of cells in culture. The present invention further providespeptides identified in these libraries, which include cell growthenhancing peptides, cell growth inhibiting peptides and peptides whichenhance or inhibit production of cellular proteins, particularlyproduction of β-toxin by Clostridium perfringens. Once the sequence of apeptide having the desired biological activity is identified, it may beproduced in large quantity (e.g., by chemical synthesis or expression ofrecombinant DNA) and formulated in a culture medium to produce thedesired effect on cultured cells. The libraries of the invention and thepeptides identified in them are particularly useful in certainlarge-scale, economical recombinant production methods.

DETAILED DESCRIPTION OF THE INVENTION

A limited diversity peptide library was constructed using conventionaltechniques for peptide synthesis and was subsequently screened forbiologically active peptides exhibiting certain desired characteristics.The initial goal was to identify peptides which could be included inculture media to increase the amount of β-toxin produced by C.perfringens, either by increasing cell growth (i.e., cell number) or byincreasing the amount of toxin produced per cell. The library of initialcandidates was based on several design criteria. First, it is known thatproteose peptone is a preferred hydrolysate for culturing C.perfringens. Proteose peptone is manufactured using pepsin, so leucinewould be one of the more common C-termini in the peptides of thehydrolysate. Accordingly, a tetrapeptide library was constructed withleucine as the C-terminus (the fourth position of the peptide) andalanine (a simple amino acid) in the third position as a spacer. The tenSelected Amino Acid Group Representatives shown below, each representinga group of related amino acids, were selected for insertion at theremaining first two positions of the peptides. Selection of the grouprepresentative amino acid is typically based on ease of peptidesynthesis using that amino acid. Selected Amino Acid Alternative GroupCluster Group Group Representative Representative Acid Glu (E) Asp (D)Amide Gln (Q) Asn (N) Hydroxy Aliphatic Ser (S) Thr (T) Small AliphaticAla (A) Beta Aliphatic Val (V) Ile (I) Large Aliphatic Leu (L) Met (M)Aromatics Phe (F) Tyr (Y) Trp (W) Basic Lys (K) Arg (R) Other Pro (P)His (H) Gly (G) Cys (C)These ten amino acids were substituted in each of the first twopositions, resulting in a limited diversity library consisting of 100different tetramer sequences. This library is referred to as the XXALlibrary, with “X” indicating an amino acid selected to represent acluster group.

Peptides for the peptide library may be synthesized by any suitablemethod known in the art, such as FMOC chemistry of Atherton and Sheppard(1989) in solid phase peptide synthesis (Merrifield, 1965). Bocchemistry may also be used as well as synthesis on a variety ofdifferent solid supports, “tea-bag” synthesis (Houghten), and split anddivide combinatorial methods. Solution phase methods for peptidesynthesis may also be used. The library peptides may includemodifications to the C-terminus (e.g., amides and esters), theN-terminus (e.g., acetyl) and non-naturally occurring amino acids (e.g.,norleucine) to assess the effect of such modifications on peptideactivity.

To identify peptides in the library which positively or negativelyaffect cell growth in culture, the library is screened in a growthassay. The selected cells are first grown in appropriate culture mediawithout peptide supplement, then subcultured in media supplemented witheach of the library peptides. The screening medium may be a complexmedium for the selected cell type, but is preferably a defined medium toallow evaluation of peptide effects without interference from undefinedmaterials present in the medium. It is also preferable to optimize thebase medium for cell growth prior to peptide screening, althoughunoptimized media may also be used. In the present work, growth of C.perfringens in the presence and absence of the peptides was evaluated ina basal medium rich in amino acids and containing the necessaryvitamins, metals, and simple carbon source. However, selection of anappropriate medium for growth screening of other cell types is routineand within the skill in the art. After an appropriate incubation time,growth of each peptide-supplemented culture is compared to growth inunsupplemented medium. The extent of growth may be evaluated using anyof the methods customarily used, including optical density (OD₆₀₀), CO₂production, O₂ consumption, ATP, fluorescence, bioluminescence, manualor automated colony counts on culture plates and impedance of anelectrical field. Screening may be performed in standard cultures or inmicrotiter plate formats.

When it is desired to identify peptides in the library which positivelyor negatively affect production of a cell product, the library isscreened in an assay appropriate for detection of that cell product.Again, the selected cells are first grown in appropriate culture mediawithout peptide supplement, then subcultured in media supplemented witheach of the library peptides. The screening medium may be a complexmedium for the selected cell type, but is preferably a defined medium toallow evaluation of peptide effects without interference from undefinedmaterials present in the medium. It is also preferable to optimize thebase medium for protein expression prior to peptide screening, althoughunoptimized media may also be used. A particular goal of the presentwork was to identify peptides which affect β-toxin production byClostridium perfringens. In this case, β-toxin secreted from the cellwas quantitated in a sandwich ELISA assay using two mouse anti-β-toxinmonoclonal antibodies followed by a goat anti-mouse IgG2A conjugated tohorse radish peroxidase (HRP). Toxin was quantitated by serial dilutionof the cultures and compared to toxin produced by cultures which did notcontain added peptide (base media cultures). Absorbance was read at 492nm and the B₅₀ values (the dilutions at which the A492 signal is 50% ofthe maximum signal) were calculated and averaged for replicate cultures.To obtain the total toxin production value the reciprocal of the B₅₀value was multiplied by the OD₆₀₀. Toxin per cell was expressed astoxin/OD. Such ELISA assay formats are easily adapted for detection ofother cell products for which monoclonal antibodies or other specificbinders or ligands are available or can be generated. In addition to thesandwich ELISA assay just described, other immunoassay formats may beemployed to quantify β-toxin or other products of interest. Theseinclude radioimmunoassay (RIA), direct ELISA, ELISA's using otherindicating enzymes, ELISA's using fluorescent reporter molecules andflow-through assays such as those which employ surface plasmon resonancedetection. In addition, in the present work the ELISA results forβ-toxin were confirmed in a bioassay. This was done to confirm that anyincrease in beta toxin detected by immunoassay represented functionaltoxin. Supernatants from peptide-supplemented cultures were diluted and0.2 mL of each dilution was inoculated into mice. Unsupplemented culturemedia served as a negative control. Mortality was recorded 24 hrs. afterinjection and the greatest dilution to produce a 50% mortality rate wasused as a measure of the amount of β-toxin produced.

Several peptides which affect cell growth were identified in the initialscreening of the XXAL library. GEAL (SEQ ID NO:1) enhanced growth of C.perfringens by about 40%, whereas KLAL (SEQ ID NO:2) inhibited growthsubstantially. SEQ ID NO:2 was so inhibitory to growth that the stage IIculture did not reach 1 OD, the minimum requirement for proceeding totesting in stage III. The constituent amino acids of SEQ ID NO:1 and SEQID NO:2 produced no significant difference in growth as compared to thebase medium alone. EKAL (SEQ ID NO:3) also substantially enhanced growthin both crude form (2×improvement in growth) and purified form(3.5×improvement in growth). ESAL (SEQ ID NO:4) was also found toenhance growth in both crude and purified form. The fact that similarresults were observed with both crude and purified peptides indicatesthat the peptide itself, and not a minor chemical involved in peptideprocessing, is responsible for the effect.

Toxin production in response to the peptides in the XXAL library wasevaluated using 15 hr. growth and two-point ELISA values. Toxin data wascollected on 75 of the 100 peptides in this library and the number ofreplicates per tetramer ranged from 1-14. Total toxin ratio wascalculated as the quotient of the total toxin derived for mediacontaining test peptide divided by the base media total toxin value. Itwas found that VNAL (SEQ ID NO:8), SNAL (SEQ ID NO:7), DKAL (SEQ IDNO:14), and NDAL (SEQ ID NO:5) increased the total toxin ratio. LSAL(SEQ ID NO:15) did not have an effect on growth, however, itsignificantly inhibited toxin production.

The XXAL limited diversity library was rationally designed based on thepredominant C-termini found on peptides in the best-performinghydrolysate for growth of the selected cell type. This concept can beextended to design of other libraries to be screened for peptidesaffecting a variety of cells. The following table illustrates the C-and/or N-termini of peptides preferred for construction of libraries tobe screened for compounds which affect the growth of cells which preferculture in the presence of hydrolysates prepared with the indicatedenzyme or chemical reagent. Reagent Library N-terminus LibraryC-terminus pepsin L, F, M, W or Y chymotrypsin F, W, Y, L, M, N or Etrypsin K or R cyanogen bromide M V8 protease D or E endoproteinaseAsp-N D enzyme (cleaves on the N- terminal side of D) Cathepsin G F, Yor W endoproteinase Lys-C K proteinase K F, Y, W, L or I papain R or Kthermolysin L, F, I, V, M or A proline peptidase A or S P hydroxylamineG N dilute acid P D iodasobenzoate W BNPS-statole W N-chlorosuccinimideW lysyl endoproteinase K endoproteinase Arg-C R asparaginylendopeptidase N

Many such defined peptide termini generated by enzymes or chemicalcleavage are known in the art and may be adapted to produce thelibraries and peptides of the present invention. As is known in the art,it should be noted that certain of the listed enzymes (e.g., trypsin,chymotrypsin, endoproteinase Lys-C, Lysyl endoproteinase and V8protease) may be inhibited when proline follows the indicated aminoacid.

Alternatively, a more diverse and larger library may be constucted byplacing the cluster group-representative “X” amino acids in allnon-C-terminal positions of the tetrapeptides (e.g., an XXXL library).Alternatively, all amino acids in a particular group (rather than justsingle amino acids which are representative of the group) may be placedin positions 1 and 2, or in all non-C-terminal positions of thetetrapeptide. If the letter Z is used to represent any one of thepossible amino acid residues such libraries would be described as, forexample, ZZAL and ZZZL. As described above, the C-terminal amino acidmay be any of the residues associated with known enzymatic or chemicalcleavage of proteins. These concepts can be even further extended tolibraries of peptides comprising more than four amino acids or tolibraries of peptides comprising termini resulting from cleavage byother enzymes or chemicals, providing even larger and more diversepeptide libraries for screening. As the library evolved from XXAL toZZAL the following peptides were found to significantly enhance cellgrowth: NDAL (SEQ ID NO:5), NNAL (SEQ ID NO:6), SNAL (SEQ ID NO:7) andVNAL (SEQ ID NO:8). In contrast, peptide KKAL (SEQ ID NO:9) inhibitedcell growth.

The peptides EKAL (SEQ ID NO:3) and DKAL (SEQ ID NO:14) are products ofa combination of these initial two library designs. A lead found in theinitial XXAL library was used to identify a lead in the ZZAL space bysimply substituting one member of an amino acid group (E) with anothermember of the same group (D), thereby reducing the screening effort.Since the C-terminus was fixed on leucine either compound could havebeen rapidly scaled up by an economical concatemer strategy.

In addition, a maximum diversity pentapeptide library in which thetermini were not fixed was constructed wherein all permutations of anamino acid sequence were represented by a single pentapeptide sequence.This allowed the number of candidate peptides to be reduced from 3.2million to 42,504. All sequences containing C, R and W were theneliminated, reducing the total set to 20,349. This was done to avoidpotentially labor-intensive syntheses which were not necessary toexploit this new library approach. Any remaining peptides that had thesame molecular formula as another peptide in the library were alsoeliminated, resulting in a final total of 19,243 unique structures.Screening of this library produced the following results. FEFVG (SEQ IDNO:16) had the second highest mean of the peptides tested for totaltoxin production (8.79) and its effect was found to be highlyreproducible over multiple experimental repetitions. Further statisticalanalysis of SEQ ID NO:16 demonstrated that its mean for total toxinproduction was statistically significantly higher than the means below7. In optimized base medium (a defined synthetic medium which does notcontain hydrolysate), SEQ ID NO:16 enhanced growth by about 40% ascompared to the optimized base medium alone, while addition of itsconstituent amino acids to the medium (F, E, V, G) increased growth onlyabout 15%. SEQ ID NO:16 increased total toxin in the two-point ELISA byabout 2.2× over total toxin production in the optimized base mediumalone, while addition of the constituent amino acids resulted in totaltoxin production approximately equivalent to the base medium alone. Itwas also found that in commercial media containing 3.5% hydrolysateblend, SEQ ID NO:16 doubled the amount of toxin produced per cell butdid not increase growth. This accounted for a near doubling of totaltoxin produced by the culture with little or no increase in cell number.This is a particularly desirable outcome for pharmaceutical companies,as the increase in toxin is obtained without the need to processadditional cell mass.

The ten pentamers with the highest mean total toxin production wereFSLLE (SEQ ID NO:17, 8.855), FEFVG (SEQ ID NO:16, 8.786611), FSFVE (SEQID NO:18, 8.727), NEYLY (SEQ ID NO:19, 8.665), FDIST (SEQ ID NO:20,8.395), NLTEL (SEQ ID NO:21, 8.321), SQLEL (SEQ ID NO:22, 8.28375),ETLNL (SEQ ID NO:23, 8.28), NQLEV (SEQ ID NO:24, 7.81) and IKLAS (SEQ IDNO:25, 7.7475). HTVEL (SEQ ID NO:26), QNDVY (SEQ ID NO:27), LPDLF (SEQID NO:28), DTHHI (SEQ ID NO:29), FVPEK (SEQ ID NO:30), GYPEV (SEQ IDNO:31), HAPAY (SEQ ID NO:32), SNGIY (SEQ ID NO:33), KFIEK (SEQ IDNO:34), MHAPP (SEQ ID NO:35), MPNNF (SEQ ID NO:36), PELME (SEQ IDNO:37), FMSTA (SEQ ID NO:38), VNVQA (SEQ ID NO:39), KFIFE (SEQ IDNO:40), PLFEQ (SEQ ID NO:41), MMELE (SEQ ID NO:42), ALFHE (SEQ IDNO:43), YEQQN (SEQ ID NO:44), GGMPG (SEQ ID NO:45), SYIME (SEQ ID NO:46)and YEYIY (SEQ ID NO:47) also increased toxin per cell above the mean,as did VDLLG (SEQ ID NO:48), DMLQT (SEQ ID NO:49), GHPVE (SEQ ID NO:50),NEGLG (SEQ ID NO:51), YENLY (SEQ ID NO:52), KPLDV (SEQ ID NO:53), DKTNG(SEQ ID NO:54), EKALE (SEQ ID NO:55), SVMEM (SEQ ID NO:56), LADTF (SEQID NO:57), KTVGI (SEQ ID NO:58), ESLQM (SEQ ID NO:59), VEFTN (SEQ IDNO:60), ELSPH (SEQ ID NO:61), TKPFF (SEQ ID NO:62), LSFIE (SEQ IDNO:63), FEFGV (SEQ ID NO:64), GDYVS (SEQ ID NO:65), ETVNF (SEQ IDNO:66). VHVYQ (SEQ ID NO:67) and NNNNN (SEQ ID NO:68) resulted in toxinproduction above the mean obtained in 0.5% hydrolysate media. YEYIG (SEQID NO:69) in 0.5% hydrolysate media produced a total toxin mean valuegreater than twice that obtained using 3.5% hydrolysate alone. PentamersAGKAH (SEQ ID NO:70), AKHSK (SEQ ID NO:71), ATNKK (SEQ ID NO:72) andADPKD (SEQ ID NO:73) also significantly inhibited growth.

Peptides identified in the XXXX library space were also found to inhibitgrowth of C. perfringens: SKKA (SEQ ID NO:10), KGLK (SEQ ID NO:11), VKKG(SEQ ID NO:12) and GLKK (SEQ ID NO:13).

The pentamer FEFVG (SEQ ID NO:16) was modified to form the hexamersEFEFVG (SEQ ID NO:74), NFEFVG (SEQ ID NO:75), FEFVGG (SEQ ID NO:76),FEFVGE (SEQ ID NO:77) and FEFVGY (SEQ ID NO:78), which produced totaltoxin values ranging from 6 to 9.7 as compared to the base media alonewhich had a mean total toxin of 3.82.

Cell growth in chemically defined media is typically slower than inhydrolysate based media. Several peptides were found to enhance growthin chemically defined base media sufficiently to equal growth intraditional hydrolysate based media. These peptides include VFTDK (SEQID NO:79), LTKVD (SEQ ID NO:80), LLPKT (SEQ ID NO:81), PLTGG (SEQ IDNO:82), GGTPV (SEQ ID NO:83), PKGTV (SEQ ID NO:84), DDDDD (SEQ IDNO:85), KLGVK (SEQ ID NO:86), TPKTL (SEQ ID NO:87), GDVTK (SEQ IDNO:88), HPAFE (SEQ ID NO:89), FFPTD (SEQ ID NO:90), VNYQA (SEQ ID NO:91)and IILEA (SEQ ID NO:92) which all produced mean growth values of 4 at 4hours. The chemically defined screening media alone had a mean of 3.2 ODfor growth at 4 hours. ESALD (SEQ ID NO:93) also enhanced growth overthe base media.

Peptides identified as having the desired properties may be produced bya variety of methods in quantities sufficient for commercial or researchuse. The peptides may be chemically or enzymatically synthesized as isknown in the art, however, more preferably the peptides are producedusing methods for expression of recombinant nucleic acids encoding thepeptides. For recombinant production, the selected peptide sequence isfirst converted to a corresponding nucleic acid sequence which encodesthe amino acid sequence of the peptide. This may be an RNA sequencewhich is subsequently translated to produce the peptide, or it may be aDNA sequence which is then cloned into an expression vector under thecontrol of a promoter which enables the transcription of the DNAsequence with subsequent translation of the mRNA. Many such methods forrecombinant production of a desired peptide or protein sequence arewell-known to the practitioner and may be applied to production of thepeptides of the invention without the exercise of inventive skill. Thepeptides may be purified, if necessary, also using standard methods forphysical, chemical and affinity separation which are well-known to thepractitioner.

It is a particularly advantageous feature of many of the peptides of theinvention that they comprise C-termini or N-termini corresponding to theC-termini and N-termini produced by enzymatic or chemical cleavage ofproteins in traditional culture media hydrolysates. This facilitatesrecombinant production of the peptides, typically in bacteria or yeast,using concatemer constructs as are known in the art. Concatemers maycontain hundreds of copies of the coding sequence. Concatemer nucleicacid constructs encoding peptides of the invention with C-termini orN-termini which are subject to enzymatic or chemical cleavage producepolypeptides comprising repeating subunits of the peptide amino acidsequence separated by convenient cleavage sites. Cleavage using theappropriate enzymatic or chemical means releases the peptide monomer.This approach to manufacture increases the yield of the desired peptideand decreases manufacturing costs. Post-expression processing issimplified due to the cleavage site which is automatically produced bycloning of the concatamer structure. For example, the peptide NDAL (SEQID NO:5) was quickly discovered using the XXAL library approach and canbe efficiently manufactured using the concatamer strategy withsubsequent cleavage using pepsin or the endopeptidase N-ASN. The growthinhibitor KKAL (SEQ ID NO:9) could also be manufactured using theconcatamer strategy and cleavage with pepsin. FSFVE (SEQ ID NO:18) couldbe expressed as a concatamer and cleaved into monomers followingexpression using the V8 protease. As a further example, HTVEL (SEQ IDNO:26) and QNDVY (SEQ ID NO:27) both enhance beta toxin accumulation inthe culture supernatant. These peptides could be expressed from the sameconcatamer minigene, cleaved by pepsin into peptides and used withoutfurther separation since both exhibit the same attribute. Such acombination minigene may also be useful when one peptide is needed tobalance another. For example, alternating a basic sequence with anacidic sequence may make the total minigene product compatible with thehost cell. Combination minigenes may also be useful production vehicleswhen multiple peptides exhibiting different attributes are desired forformulation into the same medium. In this case, all the necessarypeptides may be expressed, processed and formulated in a singleproduction process without the need to separate the individual peptides.FEFVG (SEQ ID NO:16) can be cloned as a concatamer with a nonsensesequence spacer between each peptide coding sequence to permitliberation from the concatamer. For example, the coding sequence for thenonsense peptide DEEP (SEQ ID NO:94) could flank the sequence coding forthe media enhancer peptide, and the concatamer could be cleaved with theendoproteinase Asp-N (which cuts before aspartic acid) and prolineendopeptidase (which cuts after proline). This approach requiresmultiple reagents and the spacer may need to be separated from thedesired peptide if it is not compatible with the cell culture. Nonsensespacers may also be used between peptide coding sequences in theconcatemer to generate cleavable sites and facilitate processing inotherwise non-cleavable peptide sequences.

The preferred use of the peptides of the invention is in cell culturemedia (including media for culture of cells and tissues derived fromprokaryotes and eukaryotes, vertebrates and invertebrates) to produce adesired effect on the cells. Such effects may include increased ordecreased growth rate, increased or decreased production of a cellproduct, or increased or decreased response to a substance in theenvironment (e.g., a hormone). The base culture medium to which thepeptide is added may be a chemically defined medium or a complex mediumcontaining undefined components such as fetal calf serum (FCS) or yeasthydrolysate. Chemically defined or semi-defined media are preferred, asthe peptides of the invention are most advantageously used as a meansfor reducing or eliminating performance variability due to undefinedmedia components and for reducing or eliminating animal-derivedcomponents in media used to produce pharmaceutical products.

A selected peptide is typically added to the culture medium at aconcentration from about 0.1-25 mM more preferably from about 1.0 and 12mM. However, it is within the ordinary skill in the art to determine anappropriate concentration of an inventive peptide in a selected culturemedium. Multiple peptides may be added to the culture medium to producea synergistic effect (if both have the same effect on the cells) or toproduce multiple effects (if each peptide has a different effect on thecells).

1-4. (canceled)
 5. A peptide library comprising chemically synthesizedpeptides, each of the peptides comprising an N-terminal or C-terminalamino acid associated with enzymatic or chemical cleavage of apolypeptide and one or more additional amino acids.
 6. The library ofclaim 5 wherein at least one of the additional amino acids represents achemically related group of amino acids.
 7. The library of claim 5wherein the C-terminal amino acid of the peptides is selected from thegroup consisting of D, E, F, K, L, M, N, P, R, Y and W.
 8. The libraryof claim 5 wherein the N-terminal amino acid of the peptides is selectedfrom the group consisting of A, D, F, I, L, M, S, V, Y and W.
 9. Thelibrary of claim 5 wherein the amino acid adjacent to the N-terminal orC-terminal amino acid is a spacer amino acid.
 10. The library of claim 9wherein the spacer amino acid is A.
 11. The library of claim 5 whichcomprises tetrapeptides, pentapeptides or hexapeptides. 12-18.(canceled)
 19. The library of claim 5 comprising at least two peptideshaving different biological activities. 20-29. (canceled)
 30. A peptidelibrary selected from the group consisting of: XXAL libraries, wherein“X” indicates an amino acid representative of a chemically related groupof amino acids; a) XXXL libraries, wherein “X” indicates an amino acidrepresentative of a chemically related group of amino acids; b) ZZALlibraries, wherein “Z” represents any amino acid; c) ZZZL libraries,wherein “Z” represents any amino acid, and; d) libraries wherein asingle amino acid sequence represents all permutations of the amino acidsequence. 31-32. (canceled)